Minimal Residual Disease in ALL – First Round Survey Results

1 Responders

#	First_name	Last_name
1	Julia	Alten
2	Monika	Brüggemann
3	Mireia	Camós
4	Gianni	Cazzaniga
5	Emmanuelle	Clappier
6	Conny	Eckert
7	Sarah	Elitzur
8	Nicola	Gökbuget
9	Csongor	Kiss
10	Josep Maria	Ribera
11	Nick	Short
12	Dave	Teachey
13	Jan	Trka
14	Bela	Wrench
15	Vincent	van der Velden

2 Patient population and treatment protocol

2.1 Which ALL patient group do you primarily work with?

2.2 Which clinical study group do you represent?

2.3 Which treatment protocol are your patients primarily treated under during first-line therapy?

• MD Anderson protocols - HCVAD+blina+/-INO, mini-hyper-CVD+ino+blina, blina+ponatinib
• PETHEMA ALL2019 PETHEMA ALLPh 2022
• GMALL Regstry, GMALL Evolve, GMALL BLIVEN
• HOVON-100 study protocol (if unfit: according to Hovon-117/UKALL XII study protocol (unfit arm))
• GMALL Registry younger GMALL Registry older GMALL Evolve
• AIEOP-BFM ALL 2017, Interfant-21, EsPhALL/COG, IntReALL (SR and HR)
• GRAALL/GRAAPH-2014 EWALL-INO EWALL-Ph03
• AIEOP BFM 2017
• BFM ALL-IC 2022
• ALL Together EsPhALL for Ph+ B-ALL
• AALL1731, AALL1732, AALL2331, AALL2321

• first-line: AIEOP BFM ALL 2017, ALLTogether 1 second line: IntReALL 2010 HR, ALL-REZ BFM Registry, soon IntReALL BCP 2920, HemiSmart

  We only work with the second line therapy protocols.
• INITIALL; SJALL23H; SJALL23T; Total 17
• UKALL 14
• AIEOP-BFM ALL 2017 (AIEOP-BFM ALL in general) EsPhALL/COG Interfant-21

3 Centralized vs. Local Processing of Samples

3.1 Is cytomorphology analysis performed centrally for your diagnostic samples?

3.2 Is cytomorphology analysis performed centrally for your follow-up samples?

3.3 Is the diagnostic immunophenotyping by flow cytometry performed centrally for your samples?

Category	Comment
Other	It is recommended that immunophenotyping is performed in a central lab, but it is only prtially done centrally
Other	All samples are analyzed by accredited, “matured” national central laboratories.
Other	Diagnostic flow is centralized in 11 reference laboratories

3.4 Is flow cytometric MRD analysis performed centrally for your samples?

Category	Comment
Other	MRD Flow is only done if no molecular MRD marker is available
Other	All centralized except Monza center.
Other	See reply to previous Q.
Other	Flow-MRD is centralized in 11 reference laboratories
Other	B-ALL on trial: COG approved labs. Off study local labs. Infant and T-ALL are central labs.

3.5 Is molecular MRD analysis performed centrally for your samples?

Category	Comment
Other	Ig/TC central, BCR::ABL partly central
Other	All centralized except Padua center
Other	Molecular MRD is being performed as reasearch projects by some ALLIC national team laboratories. The method is not yet available for diagnostic purposes.
Other	Molecular-MRD is centralized in 2 accredited laboratories at H. Sant Joan de Deu (Barcelona) and H. Niño Jesús (Madrid)
Other	Molecular MRD is performed in subsets of patients with B-ALL

3.6 Are central Flow Cytometry and Molecular MRD analyses performed in the same central laboratory or in separate laboratories?

Category	Comment
Other	partly overlapping
Other	Flow-MRD and molecular-MRD are performed in the same lab only in the 2 molecular reference labs. For the rest of patients, samples are sent for the corresponding Flow-MRD lab and one of the two accredited molecular-MRD laboratories

3.7 If Flow Cytometry and Molecular MRD analyses are performed in two different laboratories, how is the sample handled?

Category	Comment
Other	several vials are taken. In theory the first sample should be send to molecular MRD (except day 15) and the second/third sample to Flow Cytometry and Moelcular genetics.

4 MRD-based risk/treatment stratification during first-line treatment

4.1 What time points are defined in your protocol for performing bone marrow aspiration for remission assessment and/or risk stratification?

• End of Consolidation (2 times)
• End of Induction (2 times)
• Afrterinterphase
• After consolidation A (younger) after consolidation I (elderly)
• After consolidation B (younger) after remission induction II (elderly)
• after consolidation I
• After Consolidation I
• after each consolidation block (at count recovery)
• After HR-1’ when hematopoiesis has recovered (HR only)
• After HR-2’ when hematopoiesis has recovered (HR only)
• After HR-3’ when hematopoiesis has recovered (HR only)
• after induction during relapse treatment
• after intensification (at count recovery)
• After intenstification 1A (younger) after consolidation II (elderly)
• after phase 1 induction (at count recovery)
• after phase 2 induction (at count recovery)
• After remission induction I
• After the first three HR blocks (HR1, HR2, HR3) each if CR was not achieved ealier, or previous MRD was <1% but >/= 0.1%
• Ater intensification 1B (Younger)
• Before Consolidation I
• before stem cell transplantation during relapse treatment
• D15 (mid-induction)
• D33 (EOI)
• D78 (end-of-early-intensification, i.e. PIB)
• day +15
• Day 15 (only Flow-MRD) (all risk groups)
• Day 15 bone marrow
• Day 15 of Induction (FCM-MRD)
• Day 29, before consolidation 1 (all risk groups)
• Day 33 (End of Induction IA) (PCR-MRD; FCM-MRD in case of missing Ig/TCR Targets)
• day 33 (post induction)
• Day 50; day 65/71/78 (according to MRD d29 & d50); post-nelarabine (day 87/92/99); post-NOPHO block A, B, C; before HSCT (HR-T-cell)
• Day 57, day 99 (DS-HR)
• Day 71/78, block A1; day 92, block A1; day 99, block B1; day 113, block B1, day 120, block C1; day 134, block C1; day 176, block B2; day 197, block C2, before HSCT (HR)
• Day 78, consolidation 2 (IR-low, IR-high, DS-IR)
• Day 78/Day 92 (End of Consolidation) (PCR-MRD; FCM-MRD in case of missing Ig/TCR Targets)
• Day 8 peripheral blood
• End early consolidation
• End induction
• End late consoloidation or Month 3 after alloHSCT
• End of consolidation, day +252
• end of Consolidation, day +78/+92
• End of cycle 4, day +84
• end of Induction, day +33
• End of induction, day+21
• Every 6 monys for the second year (chemotheraoy or alloHSCT)
• Every three months after late consolidation of ater alloHSCT (1st year)
• Mid induction
• Some trials have additional timepoints but it varies significantly
• week 12 (in non-SR patients)

4.2 What are the relevant cut-off values used for risk stratification?

5 Postremission MRD monitoring for detection of an MRD relapse

5.1 In which patients do you typically perform bone marrow aspiration for an early detection of an MRD relapse?

Category	Comment
Other	Not systematically performed
Other	before and after allo-SCT, after relapse, after CART
Other	We follow the protocol. Additionally, in case of relapse suspicion based on clinical or laboratory findings
Other	Before and after allo-SCT
Other	all because all are relapses

5.2 How many times do you perform bone marrow aspiration for relapse detection according to your protocol?

Category	Comment
Other	depends on a situation, can be more than four times, follow up post-SCT is usually day30, day 60, day 180, 1 yr, 2yr, 3yr
Other	Never. Transplant is not included on our trials.
Other	currentl not defined, very frequent
Other	within the first year after each block
Other	depending of the MRD-kinetics and the risk profile (3-5 x in HR without alloHSCT and 8-10 x in pts with alloHSCT)

5.3 Do you conduct additional MRD assessments based on individual clinical decisions?

5.4 At what intervals per patient do you perform post-remission MRD assessment for detection of an MRD relapse?

Category	Comment
Clinical Signs	prolonged cytopenias
Clinical Signs	Standard risk (after TP1)/Medium risk patients (after TP2): Only if there are clincal or laboratory signs of relapse (e.g. prolonged cytopenia…)
Fixed Intervals	Every 3-4 months
Fixed Intervals	evry three months at 1st year, every six months for the 2nd year and then yearly up to 5 years
Fixed Intervals	Ph negative ALL, chemotherapy: first year: prior to each consolidation cycle, second+third year: every 3 months, fourth year: evey 6 months. Ph negative ALL, after SCT: every 3 months. Ph+ ALL, after SCT: every 6 weeks in blood, every 3 months in BM.
Fixed Intervals	every 3 months (three years)/ 2 months (2 years), and if there are clinical signs of relapse
Fixed Intervals	3
Fixed Intervals	depends on a situation, follow up post-SCT is usually day 30, day 60, day 180, 1 yr, 2yr, 3yr, in relapsed based on relapse protocol
Fixed Intervals	end of therapy
Fixed Intervals	MRD post alloHSCT (d+30, d+60, d+100, d+180, d+365)
Other	If there are clinical/lab signs suspicious for relapse, and in pts. after alloHSCT
Other	after each treatment block, ad hoc practice in maintenance every 3 months
Other	MRD after each element of HR-Therapy (AIEOP-BFM:all HR-patients: TPHR1, TPHR2, TPHR3 (=MRD after each HR-Block), additionally all HR-T-ALL, all Infants, B-ALL (not atl east 2 x negative in subsequent time points): prior to Re-Induction (prior to 2. Protocol III and 3. Protocol III)

5.5 Until when do you perform bone marrow aspiration for relapse detection?

Category	Comment
Dx Months	~36 months
Dx Months	until end of year 4 (chemotherapy patients)
Dx Months	uptill 5 years after reaching CR
Dx Months	approximately 36-48 months
Dx Months	x1 at end of therapy
Dx Months	end of intensive chemotherapy (see question 16)
Transplant Months	Not fully standardized. Depends on MRD kinetics.
Transplant Months	approximately 36 months
Transplant Months	see above, in negative samples post-SCT last BM is 3 years post-SCT, after that yearly PB. But depends on the clinical situation and individual decision of physicians
Transplant Months	1 year
Lifelong	evry three months at 1st year, every six months for the 2nd year and then yearly up to 5 years
Other	evry three months at 1st year, every six months for the 2nd year and then yearly up to 5 years

5.6 What are the relevant MRD cut-off values used for relapse detection in Ph-negative ALL?

Other Responses for MRD_cutoff_PhNeg_other

Response	Answer
Other	Positivity below QR: warning signal, MRD >1XE-04: molecular relapse
Other	5x10-2
Other	any MRD positivity, but in positive not quantifiable verified by NGS
Other	Any MRD-positivity with further confirmation with other method and different sample
Other	We follow PDL guidelines
Other	For relapse definition more than 1 method is needed for confirmation, if MRD is < 5%. MRD of 5E-04 is highly suspicious of pending relapse if detected late in therapy

5.7 What are the relevant IG/TR-based MRD cut-off values used for relapse detection in Ph-positive ALL?

Other Responses for MRD_cutoff_PhPos_other

Response	Answer
Other	not fully standardized, probably like in Ph-negative ALL.
Other	5x10-2
Other	we do BCR/ABL on DNA level simultaneously and solve individually (CML-like cases)
Other	Any MRD-positivity with further confirmation with other method and different sample
Other	We follow PDL guidelines
Other	do not use IG/TR-MRD; 1x10-2 for flow
Other	see question 18

5.8 What are the relevant BCR::ABL1-based MRD cut-off values used for relapse detection in Ph-positive ALL?

Other Responses for MRD_cutoff_BCRABL_other

Response	Answer
Other	Likely not relevant with IG/TR testing
Other	none
Other	reported in the context of simultaneous measurement of DNA-based BCR/ABL and Ig/TR (e.g. BCR/ABL 1E-02 and Ig/TR negative does not need to be relapse in CML-like cases)
Other	ALLIC group did not define MRD cut-off value for diagnosing molecular relapse in Ph+ALL. LOQ in CML is 1e-05
Other	Any MRD-positivity with further confirmation with other method and different sample
Other	We follow PDL guidelines
Other	BCR::ABL1 positivity without Ig/TCR positivity and zytomorphological absence of blasts is not considered a relapse (this constellation is suspicious for misdiagnosis in the beginning –> CML in blasts crisis )

6 Relapse confirmation

6.1 Is a conversion to MRD-positivity considered equivalent to relapse?

Category	Comment
Yes, always	Yes if converts from negative to positive but only if there is high confidence that the finding reflects true MRD (e.g. not “background”), ideally confirmed on a follow-up sample.
Dependes on MRD level	If ther is higher than 1x10e-4, confirmed in a second sample
Dependes on MRD level	1xE-04
Dependes on MRD level	if >0,01%
Dependes on MRD level	10-4 but this is defined then as molecular relapse which is different from hematologic relapse
Dependes on MRD level	Clearly if 1E-02, but it would trigger clinical actions if 1E-04 if confirmed by 2 tecniques in 2 different consecutive samples
Dependes on MRD level	1x10-2
Dependes on MRD level	one log increase required
Dependes on MRD level	MRD 1 -<5%: two other tests with >=1% or second BM. MRD <25% one additional test >= 1%. doi: 10.1182/blood.2021012328. PMID: 34192312; PMCID: PMC8952186.
No, never	depends on ALL type-different situation in CML-like ALL, see above. In Ph-neg ALL pos NQ after negative always verified by NGS
No, never	Only if MRD exceedes the 1% (1–02) limit for CR as defined by the P-d-L paper
Depends on additional findings	MRD-positivity conversion should be unambiguous: either high level, or if low, without any doubt (confirmed on a second sample or a second marker or technique, or with clinical signs of extramedullary relapse))
Depends on additional findings	Flow or/and morphology

6.2 Should the conversion from IG/TR-based MRD-negativity to MRD-positivity be confirmed by a consecutive sample?

Other Responses for MRD_confirm_IGTR_other

Response	Answer
Other	depends on the context and MRD level
Other	We follow PdL so depends on if a confirmatory test and level of detection
Other	depends on the level <10-4 likely requires repeat
Other	No, a single positive result is sufficient: depends on the Level (see 21)

6.3 Should the conversion from BCR::ABL1-based MRD-negativity to MRD-positivity be confirmed by a consecutive sample?

Other Responses for MRD_confirm_BCRABL_other

Response	Answer
Other	Only if accompanied by MRD by IG/TR
Other	IG/TR to be analyzed
Other	Relapse should be confirmed by IG/TR MRD in bone marrow
Other	in CML like completely different situation, usually repeated sample necessary
Other	We follow PdL
Other	no routine BCR::ABL1 monitoring performed

6.4 If MRD conversion requires confirmation, within what time frame should the consecutive sample be taken?

Category	Comment
Other	No clear rule
Other	2 weeks
Other	We follow PdL
Other	<1 week for positive >1% without morphology, 2-4 weeks for lower level

7 Clinical consequences of postremission MRD reoccurence

7.1 What are the clinical consequences when MRD recurrence is detected during first-line chemotherapy?

Other Responses for MRD_recur_1L_other

Response	Answer
Other	it depends on MRD levels, frequently, a further BM is planned to monitor increase
Other	No defined rule, to be discussed
Other	depends on the protocol, usually SCT with salvage before
Other	It depends on the MRD level, time point and the clinical setting
Other	If meets PdL criteria for relapse we treat as a relapse
Other	answers depend of the level of MRD positivity: >=5E-04 after TP2 (day 78/92) Indication for alloHSCT and intensification of therapy. Below 5E-4: Obersevation without immediate intervention

7.2 What are the clinical consequences when MRD recurrence is detected after the end of first-line chemotherapy?

Other Responses for MRD_recur_post_1L_other

Response	Answer
Other	depending on CR time, Relapse within 1 year: salvage; if CR>1 year first line induction rretarted
Other	A further BM is planned to monitor increase
Other	No defined rule, to be discussed
Other	not measured in non-HR patients, in HR patients is defined by protocol
Other	It depends on the MRD level, time point and the clinical setting
Other	If meets PdL criteria for relapse we treat as a relapse
Other	it depends on the MRD level see question 25

7.3 What are the clinical consequences when MRD recurrence is detected after stem cell transplantation?

Other Responses for MRD_recur_post_SCT_other

Response	Answer
Other	not fully standardized
Other	if no GVHD: reduce immune suppression. Otherwise DLI or blina
Other	reduction of immunosuppression; short term confirmation of MRD increase on another BM sample
Other	based on the level-low MRD IST tapering, DLI, in high level CART, 2nd SCT..
Other	Individual decision made by the transplant team
Other	It depends on the MRD level, time point and the clinical setting
Other	Depends on type of transplant, availability of DLI, ability to add a targeted or immunotherapeutic agent
Other	it depends on the MRD level. Low positivity: reduction of Immunosuppression if possible, DLI

8 Bone marrow sampling

8.1 In the clinical study your patients are treated in, is there a standard operation procedure (SOP) for bone marrow sampling?

Category	Comment
Other	There is a manual that states: 4.6.3 Material  10 ml Knochenmark und/oder 10 ml peripheres Blut  Von jedem Untersuchungszeitpunkt, möglichst 1. Aspirat  EDTA bevorzugt, Heparin auch möglich Die Zeitpunkte der Remissionskontrollen und Einsendung von Probenmaterial zur MRD-Untersuchung sind den aktuellen Protokollen bzw. Therapieempfehlungen zu entnehmen. Praktisch wird die KM-Punktion jeweils nach Regeneration durchgeführt. Es handelt sich also nicht um Aplasie-Punktionen. In addition, we hav a local SOP in Kiel
Other	instructions are give in an extra document of the sample request form

8.2 Apart from its use in diagnosing and classifying ALL, how else is diagnostic bone marrow aspirate utilized in clinical or research settings?

Other Responses for BM_use_other

Response	Answer
Other	research on add-on projects
Other	For pilot research purposes
Other	molecular genetics

8.3 Which pull of the bone marrow aspiration is used for MRD detection during follow-up?

8.4 If the first pull of bone marrow aspiration is not used for MRD assessment, what is it typically used for?

8.5 What collection system do you use for bone marrow aspiration?

Other Responses for BM_collection_other

Response	Answer
Other	See Kiel SOP
Other	Don’t know if it is standardized
Other	do not know and I am afraid to go ask somewhere because it seems impossible to save the answers and I would loose too much work
Other	systems differ between clinical departments

8.6 What is the volume of the collection system you use for bone marrow aspiration?

Other Responses for BM_volume_other

Response	Answer
Other	not applicable
Other	2-5 ml left and 2-5 ml right
Other	depending of age and collection system all of the answers above

8.7 What volume of bone marrow do you typically aspirate during the procedure?

Other Responses for BM_aspirate_other

Response	Answer
Other	in multiple aspirates
Other	Following the SOP , we perform 2 aspirates of 4ml (1st aspirate) and 10ml (2nd aspirate at different angle through same skin puncture)
Other	we do repeat pulls of approximately 2ccs with reanchoring
Other	depending of age and collection system all of the answers above

8.8 What volume of bone marrow do you typically provide for MRD analysis?

Category	Comment
Other	we receive DNA already extracted
Other	Optimally 2.0 mL
Other	depends on the cell number
6–10 mL	depending of age and collection system all of the answers above

9 Trephine biopsy

9.1 Do you perform bone marrow trephine biopsy?

9.2 What is your typical approach in cases of a dry tap during bone marrow aspiration?

10 Transport times

10.1 What is the typical transport time for bone marrow samples used for molecular MRD analysis?

10.2 What is the typical transport time for bone marrow samples used in flow cytometry MRD analysis?

11 Sample preparation

11.1 Are your bone marrow samples being checked for hemodilution?

Category	Comment
No, never	We send the sample to the central lab for FCM and there check for hemodilution
Yes, using Cytomorphology	Presence/absence of bone marrow particles
Yes, using Cytomorphology	assessment of spicules
Yes, using Cytomorphology	Important for remission evaluation at day 33 (TP1)
Yes, using Flow cytometry	checked for but no calculation, remark made in conclusion
Yes, using Flow cytometry	do not know and do not want to go ask FC guys because it seems impossible to save the answers in the process. can send later
Yes, using Flow cytometry	Presence of <2% of EBL
Yes, using Flow cytometry	Only day 15: Important for FCM-MRD evaluation

11.2 If your bone marrow samples are assessed for hemodilution, how do you utilize the obtained information?

• only remark with warning in conclusion
• The test is performed anyway, final evaluation with the physician
• A new sample is performed in case of hemodilution
• write a comment to the results
• If high-risk criteria are fulfilled (depending on the TP), resuts will be released for clinical used but withhold by the lab below those limits.
• A comment is detailed in the cytomorphology report.
• We might repeat depending on how much the result impacts clinical care.
• Generally still used but with note made of hemodilution
• Hemodilution is mostly relevant fror Response Evaluation at day 33 (TP1). If cytomorphology showes signs of heodilution and therefore a not representative BM, the BM puncture should be repeated. However we always measure MRD in both samples and use the higher value for stratification, because unsually MRD-levels are decreasing even without continuation of therapy after Day 33.

11.3 Are your bone marrow samples being processed using Ficoll separation prior to MRD analysis?

11.4 When preparing the first dilution step (typically 1E-01) for the standard curve from a Ficoll-processed sample, do you take the blast percentage into account?

Category	Comment
Other	yes, when blasts are below 50%
Other	yes if it is below 50%
Other	this varies based on each center practice
Other	we adjust the standard curve according to cytomorphology, if blast infiltration is below 50% however we are trying to implement Flow after Ficoll at the moment in order to be more exact

11.5 Is flow cytometry analysis performed before or after Ficoll separation?

Category	Comment
Other	I do not know
Other	and after for defining blast percentage for molecular standard curve
Other	before, in some situations (low number of blasts, sorting for RNAseq) later
Other	The complete Flow cytometry analysis is performed before Ficoll separation. After Ficoll separation we perform an additional tube to calculate the blast percentage post-Ficoll
Other	planned: after ficoll

11.6 Is cytomorphology analysis performed before or after Ficoll separation?

Category	Comment
Other	not performed
Other	cytomorphology is performed on BM smears and not in the same sample

12 MRD assesment methods

12.1 Which method(s) do you employ for MRD assesment in Ph-neg, KMT2A-neg BCP-ALL?

12.2 Which method(s) do you employ for MRD assesment in Ph-positive BCP-ALL?

12.3 Which method(s) do you employ for MRD assesment in KMT2A-positive BCP-ALL?

12.4 Which method(s) do you employ for MRD assesment in T-ALL?

12.5 Do you use any molecular markers for MRD analysis other than those mentioned above?

Category	Comment
Other	depends on target, number of classical targets
Other	no, not at the moment
Other	If flow unavailable
Other	SIL-TAL and IKZf1

12.6 Which additional molecular markers do you use for MRD analysis?

13 Technical requirements flow cytometry

13.1 Which anticoagulant do you use for bone marrow samples intended for flow-cytometry MRD analysis?

13.2 Do you apply bulk lysis to your samples?

Category	Comment
NA	I do not know
Only at certain time points (please specify)	Not in aplasia
Only at certain time points (please specify)	in case of low cellularity
NA	Sometimes
Only at certain time points (please specify)	only remission samples (day 33 onwards), not day 15 and not inital diagnosis

13.3 What is the minimum number of cells you typically measure?

Category	Comment
Other	I do not know ( I am clinician)
Other	Depends on the time-point. In ALL-BFM, we do day 15 MRD, and there we acquire 500,000 cells. In GMALL and in ALL-BFM at other time-points, we aim for 4,000,000 events.
Other	Dx 100,000, day 15 100,000-500,000, day 33 more than 1,000,000
Other	500,000 cells
Other	Varies based on lab and method
Other	remission spamples:1-10. Mio, Initial + day 15 : 100.000-500,000

13.4 What is the minimum number of events required to define MRD positivity (LOD)?

Category	Comment
Other	do not know ( I am clinician)
Other	10 events
Other	Varies based on lab and method

13.5 What is the minimum number of events required to quantify MRD (LOQ)?

Category	Comment
NA	do not know ( I am clinician)
Other	40 events
Other	Varies based on lab and method

13.6 What level of sensitivity (LOD) is typically achieved in your MRD analysis?

Other Responses for FC_LOD_other

Response	Answer
Other	BCP-ALL: E-05, T-ALL: rather E-04 or less
Other	depends on timepoint, see above
Other	2X10-5
Other	It varies according to the time point
Other	day 15: 1E-03 (without bulk lyisis)

13.7 What level of quantification (LOQ) is typically achieved in your MRD analysis?

Other Responses for FC_LOQ_other

Response	Answer
Other	BCP-ALL: 5xE-05, T-ALL: rather E-04 or less
Other	day 15 0.1%, day 33 0.1%-0.01%
Other	6X10-5
Other	It varies according to the time point

13.8 Do you use a commercial assay or an in-house assay for your MRD analysis?

Category	Comment
Commercial	NSG MRD is commercial
Commercial	ClonoSeq for NGS-based
EuroFlow	BCP-ALL: EuroFlow, but as in-house assay. T-ALL: in-house assay according to BFM-Flow SOP
EuroFlow	BCP-ALL MRD assay
EuroFlow	BD CytognosTM B-cell Precursors ALL MRD
EuroFlow	B-MRD plus 1 additional in-house tube
In-house	I-BFM standard
In-house	Flow is both performed locally and centrally and depends on population and trial and timepoints
In-house	St. Jude in house
In-house	T-MRD

14 Technical requirements IG/TR qPCR

14.1 Which anticoagulant do you use for bone marrow samples intended for IG/TR-based qPCR MRD analysis?

14.2 What is the minimum DNA amount you typically analyze per reaction?

## No comments available.

14.3 What level of sensitivity is typically achieved in your MRD analysis?

14.4 What quantitative range is typically achieved in your MRD analysis?

14.5 How do you report results that are above the sensitivity threshold but below the quantitative range?

15 Technical Requirements IG/TR ddPCR

15.1 Which anticoagulant do you use for bone marrow samples intended for IG/TR-based ddPCR MRD analysis?

15.2 What is the minimum DNA amount you typically analyze per molecular target?

## No comments available.

15.3 What is the minimum number of events required to define MRD positivity?

## No comments available.

15.4 What level of sensitivity is typically achieved in your MRD analysis?

15.5 Do you account for background noise from healthy cells in your MRD analysis?

15.6 Do you use a commercial assay or an in-house assay for your MRD analysis?

## No comments available.

16 Technical Requirements IG/TR amplicon NGS

16.1 Which anticoagulant do you use for bone marrow samples intended for IG/TR-based NGS MRD analysis?

16.2 What is the minimum DNA amount you typically analyze per sequencing library reacion?

Category	Comment
Other	This is centrally done and we are not privy to the amount needed

16.3 What is the minimum number of reads required to define MRD positivity?

Category	Comment
Other	This is proprietary and we are not provided it

16.4 What level of sensitivity is typically achieved in your MRD analysis?

16.5 What quantitative range is typically achieved in your MRD analysis?

16.6 Do you account for background noise from healthy cells in your MRD analysis?

16.7 Do you use a commercial assay or an in-house assay for your MRD analysis?

Category	Comment
Commercial	clonoSEQ
Commercial	Clonoseq

17 Technical requirements KMT2A qPCR

17.1 Which anticoagulant do you use for bone marrow samples intended for KMT2A-based qPCR MRD analysis?

17.2 What is the minimum DNA amount you typically analyze per reaction?

## No comments available.

17.3 What level of sensitivity is typically achieved in your MRD analysis?

17.4 What quantitative range is typically achieved in your MRD analysis?

17.5 Do you account for background noise from healthy cells in your MRD analysis?

17.6 Do you use a commercial assay or an in-house assay for your MRD analysis?

Category	Comment
In-house	RT-PCR; note, we use RNA rather than DNA
In-house	patient-specific assays

18 Technical Requirements KMT2A ddPCR

no responses

19 Technical Requirements DNA-based BCR::ABL1 qPCR

19.1 Which anticoagulant do you use for bone marrow samples intended for BCR::ABL1-based qPCR MRD analysis?

19.2 Which housekeeping gene do you use for BCR::ABL1 qPCR normalization?

19.3 How many copies of the housekeeping gene do you typically aim for in BCR::ABL1 transcript quantification?

Category	Comment
Other	depends on situation (we use GUSB)
NA	not applicable for DNA-based analysis

19.4 How do you quantify the housekeeping gene as the denominator for BCR::ABL1 MRD assessment:

Category	Comment
NA	not applicable for DNA-based analysis

19.5 What is the source of the plasmids you use for BCR::ABL1 quantification?

19.6 What is the minimum number of positive replicates required to define MRD positivity?

Category	Comment
NA	not applicable for DNA-based analysis

19.7 How do you report the MRD value for the major BCR::ABL1 transcripts?

19.8 How do you report the MRD value for the minor BCR::ABL1 transcripts?

19.9 Do you use the CML MRD nomenclature to interpret BCR::ABL1-MRD results in Ph+ ALL (MMR, MR4, MR4.5, MR5)?

Category	Comment
NA	not applicable for DNA-based analysis

19.10 Do you use a commercial assay or an in-house assay for major BCR::ABL1-transcript-based MRD analysis?

Category	Comment
In-house	patient-specific assay

19.11 Do you use a commercial assay or an in-house assay for minor BCR::ABL1-transcript-based MRD analysis?

Category	Comment
In-house	patient-specific assay

20 Technical Requirements RNA-based BCR::ABL1 qPCR

20.1 Which anticoagulant do you use for bone marrow samples intended for BCR::ABL1-based qPCR MRD analysis?

20.2 Which housekeeping gene do you use for BCR::ABL1 qPCR normalization?

20.3 How many copies of the housekeeping gene do you typically aim for in BCR::ABL1 transcript quantification?

Category	Comment
NA	do not know ( I am clinician)
Other	aim for >100,000 ABL1 copies
Other	NA (Cepheid)
Other	depends on situation, in poor samples we use bad quality samples and comment
Other	15-35 copies GAPDH

20.4 How do you quantify the housekeeping gene as the denominator for BCR::ABL1 MRD assessment:

Category	Comment
NA	do not know ( I am clinician)
Other	cepheid

20.5 What is the source of the plasmids you use for BCR::ABL1 quantification?

20.6 How do you report the MRD value for the major BCR::ABL1 transcripts?

20.7 What is the minimum number of positive replicates required to define MRD positivity?

Category	Comment
NA	do not know ( I am clinician)

20.8 How do you report the MRD value for the minor BCR::ABL1 transcripts?

20.9 Do you use the CML MRD nomenclature to interpret BCR::ABL1-MRD results in Ph+ ALL (MMR, MR4, MR4.5, MR5)?

Category	Comment
NA	do not know ( I am clinician)
Other	Only if a clinical trial asks us to do so

20.10 Do you use a commercial assay or an in-house assay for major BCR::ABL1-transcript-based MRD analysis?

Category	Comment
Commercial	Cepheid
Commercial	Qiagene
Commercial	Xpert BCR-ABL ULTRA (Cepheid)
Commercial	commercial for CML
In-house	IRMM ERM-AD623 Plasmid and in-house assay
In-house	in house for MRD in Ph+ ALL

20.11 Do you use a commercial assay or an in-house assay for minor BCR::ABL1-transcript-based MRD analysis?

Category	Comment
Commercial	Ipsogen BCR::ABL1 m-bcr Kit
Commercial	Cepheid
Commercial	Qiagene
Commercial	commercial for CML
In-house	in house for MRD in Ph+ ALL

21 Technical Requirements DNA-based BCR::ABL1 ddPCR

no responses

22 Technical Requirements RNA-based BCR::ABL1 ddPCR

no responses

23 Technical requirements STIL::TAL1 qPCR

23.1 Which anticoagulant do you use for bone marrow samples intended for STIL::TAL1-based qPCR MRD analysis?

23.2 What is the minimum DNA amount you typically analyze?

## No comments available.

23.3 What level of sensitivity is typically achieved in your MRD analysis?

23.4 What quantitative range is typically achieved in your MRD analysis?

23.5 Do you account for background noise from healthy cells in your MRD analysis?

23.6 Do you use a commercial assay or an in-house assay for your MRD analysis?

## No comments available.

24 Technical requirements STIL::TAL1 ddPCR

no responses

25 Molecular MRD markers

25.1 How do you manage patients who do not have a suitable molecular MRD marker?

26 Prioritization in cases with multiple MRD results

26.1 If both BCR::ABL1-based MRD on a DNA-level and IG/TR-based MRD results are available, which one do you prioritize for reporting?

Category	Comment
Other	do not use DNA for BCR-ABL
Other	only IG/TR performed
Other	We do not perform BCR::ABL1-based DNA MRD. However, in case such result was available, both results would be reported from the lab to the clinicians. The concordance or discordance of results and final conclusions are discussed in a multidisciplinary meeting for every MRD assessment for all patients
Other	do not use IG/TR-MRD
Other	BCR::ABL1 is not used for stratification

26.2 If both BCR::ABL1-based MRD on an RNA-level and IG/TR-based MRD results are available, which one do you prioritize for reporting?

Category	Comment
Other	Both results are reported from the lab to the clinicians. The concordance or discordance of results and final conclusions are discussed in a multidisciplinary meeting for every MRD assessment for all patients. However, in the EsPhALL trial only the IG-TR MRD results are considered.
Other	do not use IG/TR-MRD
IG/TR-based MRD result	BCR::ABL1 is not used for stratification

26.3 If both KMT2A-based MRD and IG/TR-based MRD results are available, which one do you prioritize for reporting?

Category	Comment
Other	We do not perform KMT2A-based MRD. However, in case such result was available, both results would be reported from the lab to the clinicians. The concordance or discordance of results and final conclusions are discussed in a multidisciplinary meeting for every MRD assessment for all patients. Generally speaking, we would consider the higher MRD value
Other	do not use IG/TR-MRD

26.4 In switch ALL cases, which MRD method do you prioritize for reporting?

Category	Comment
Other	both results with comment from FC group
Other	Both results are reported from the lab to the clinicians. The concordance or discordance of results and final conclusions are discussed in a multidisciplinary meeting for every MRD assessment for all patients. In switch ALL cases, we usually find higher MRD values with IG/TR and consider this higher MRD result for taking clinical actions.
Other	in the US all results have to be reported if a clinical test. Protocols can give guidance and local institutions have their own standard. But it is not allowed for the lab who performs the assay to prioritize

27 MRD vs. cytomorphology for remission assessment

MRD and cytomorphology results are not always concordant. In the following situations, which value do you choose for clinical reporting to state the blast percentage at the time of remission assessment?

27.1 Cytomorphology >25% and:

27.1.1 MRD <1%

27.1.2 MRD 1-5%

27.1.3 MRD 5-25%

27.1.4 MRD >25%

27.2 Cytomorphology 5-25% and:

27.2.1 MRD <1%

27.2.2 MRD 1-5%

27.2.3 MRD 5-25%

27.2.4 MRD >25%

27.3 Cytomorphology <5% and:

27.3.1 MRD <1%

27.3.2 MRD 1-5%

27.3.3 MRD 5-25%

27.3.4 MRD >25%

28 MRD vs. cytomorphology for relapse assessment

MRD and cytomorphology results are not always concordant. In the following situations, which value do you choose for clinical reporting to state the blast percentage at the time of relapse assessment?

28.1 Cytomorphology >25% and:

28.1.1 MRD <1%

28.1.2 MRD 1-5%

28.1.3 MRD 5-25%

28.1.4 MRD >25%

28.2 Cytomorphology 5-25% and:

28.2.1 MRD <1%

28.2.2 MRD 1-5%

28.2.3 MRD 5-25%

28.2.4 MRD >25%

28.3 Cytomorphology <5% and:

28.3.1 MRD <1%

28.3.2 MRD 1-5%

28.3.3 MRD 5-25%

28.3.4 MRD >25%

29 CSF MDD

29.1 Do you perform MDD assessment in cerebrospinal fluid?

Category	Comment
Other	we sometimes receive CSF, not systematically recommended
Other	if asked and sent by clinicians
Other	rarely to confirm antigen expression following identified CNS involvement
Other	only in case of suspected relapse

29.2 If MDD is detected in cerebrospinal fluid, are there any therapeutic consequences?

Category	Comment
Other	only CNS3
Other	No, but performing flow-cytometry assessment is strongly recommended by the protocol to confirm the presence of blasts identified by cytomorphology
Other	not sure, not standardized

30 CSF MRD

30.1 Do you perform MRD assessment in cerebrospinal fluid?

Category	Comment
Other	we sometimes receive SCF, but it is not systematically recommended
Other	if asked and sent by clinicians
Other	not sure, not standardized
Other	rarely to confirm antigen expression following identified CNS involvement
Other	only in case of suspected CNS-relapse/persisting blasts in cytospin

30.2 If MRD is detected in cerebrospinal fluid, are there any therapeutic consequences?

Category	Comment
Other	if MRD+ switch of therapy
Other	Each assessment is discussed in a joint meeting of the laboratories and the clinical teams

31 CSF MRD Methods

31.1 Which method do you use for MRD assessment in cerebrospinal fluid?

31.2 How is MRD positivity in cerebrospinal fluid defined when detected using IG/TR qPCR?

Category	Comment
Other (please comment)	We do not use IG/TR qPCR
Other (please comment)	We do not perform IG/TR MRD in CSF
According to CSF-specific rules (please comment)	not standardized yet

31.3 What is the minimum number of events required to define MRD positivity in cerebrospinal fluid by flow cytometry?

Category	Comment
NA	do not know ( I am clinician)

32 Additional topics

32.1 In your opinion, are there any important topics we have missed in this survey? If yes, please specify in the comments.

Category	Comment
No	I cannot successfully answer to many technical comments, as I am a clinician
Yes	some questions are difficult to answer, this answers would be much more complicated.
No	level of control gene in DNA-based methods (which is acceptable)-there are transcript copies.
Yes	Communication between the different labs and between the lab and the clinical teams is crucial to discuss both the concordant and the discordant results. We perform an integrated biological report at diagnosis and at each MRD time point, and each BM assessment is discussed weekly with the clinicians.

A possible topic to discuss is the presence of integrated reports and the (fluid)communication between clinicians and laboratory staff.

Comment for questions 148-153: both the cytomorphology and the MRD results are reported from the lab, and any discordances are studied and discussed in a meeting with the lab and clinical teams to try to explain the discordance (hemodilution of the sample, regenerative BM with a high percentage of B-cell normal precursors (hematogonies), difficult LAIPs, etc) and agree on a conclusion on the results. | |Yes |planned / expected changed in the near future | |Yes |diagnostic procedures and laboratory practices will be country specific, in the UK we operate via the SIHMDS model. For instance, it is not our practice to regress or censor multimodal results for remission to one output, all our reported out to the requesting clinician. In addition practices are guided by the treatment approval criteria - e.g. flow based assessments for CAR-T indication but molecular MRD (plus flow) for Blina for instance, so no singular system exists addressing all the clinically actionable outcomes of MRD testing |